Calf serum

INS-1E cells (Merglen et al., 2004 (link)) (kind gift of Bruno Guigas, Leiden University Medical Center, The Netherlands) were cultured in Roswell Park Memorial Institute (RPMI) medium 1640 (Gibco, Life Technologies) supplemented with 10% fetal calf serum (FCS) (GE Healthcare Life Sciences), 10 mM HEPES pH 7.4, 1 mM sodium pyruvate (Gibco, Life Technologies), 55 μM β-mercaptoethanol (Gibco, Life Technologies), 11 mM glucose (Sigma-Aldrich) and 1% (v/v) penicillin/streptomycin (Sigma-Aldrich). The human insulinoma cell line EndoC-βH1 cells (Ravassard et al., 2011 (link)) obtained from Univercell-Biosolutions, was cultured in complete medium [low glucose DMEM (Gibco, 1 g/l glucose), 2% albumin (Sanquin Bloodbank, The Netherlands), 10 mM nicotinamide (prepared by the Leiden University Medical Center pharmacy), 5.5 g/ml human transferrin (Sigma-Aldrich), 0.5 mg/ml selenite (Sigma-Aldrich), 10 ml penicillin (100 U/ml)/streptomycin (100 g/ml), with fresh β-mercaptoethanol (Sigma-Aldrich) added when culturing (to a final concentration of 0.05 mM)] in a humidified atmosphere with 5% CO₂. HeLa cells were cultured in DMEM (Lonza) supplemented with 10% fetal calf serum (FCS) (GE Healthcare Life Sciences) and 1% (v/v) penicillin/streptomycin. All cells were routinely checked for mycoplasma contamination using the MycoAlert™ Mycoplasma Detection Kit (Lonza).

To simulate knee motion and loadings, a wear simulator (model KS2-6-1000, Advanced Mechanical Technology Inc., Watertown, MA, USA) was used (Figure 2). In addition to the three wear stations, a reference station was available for soak control, which was only axially loaded. In each group, three spacer systems were used for wear evaluation and one additional spacer served as a soak control to account for liquid absorption of the cement. The wear tests were performed in force control according to ISO 14243-1:2009, with a 50% reduced axial force to consider the partial weight bearing of the patient. The simulations were carried out at 37 ± 1 °C in closed chambers filled with calf serum (PAA Laboratories GmbH, Pasching, Austria). The serum had a protein concentration of 20 g/L [17 ], and 1.85 g/L of sodium azide (NaN3) and 7.44 g/L of ethylene diamine tetraacetic acid (EDTA) were added to the calf serum to retard bacterial growth and to prevent the formation of calcium phosphate films on the surface. Tests were run at a frequency of 1 Hz for a total of 500,000 cycles. This number of loading cycles was chosen because it represents a duration of three to six months of clinical use [18 (link)].

The human cervix epidermoid carcinoma cell line HeLa was cultured at 37 °C in DMEM (Invitrogen/Life Technologies, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA). src^ts-NRK cells, rat kidney cells that were infected with ts25, a T-class mutant of Rous sarcoma virus Prague strain, were cultured at permissive temperature (32 °C) in MEM (Sigma-Aldrich), supplemented with 10% calf serum (PAA Laboratories, Pashing, Austria). Each cell line was seeded into a 96-well plate (HeLa, 4 × 10³ cells/well; src^ts-NRK, 1 × 10⁴ cells/well) and then exposed to test compounds. After 48 h incubation, cell proliferation was determined using a Cell Count Reagent SF (Nacalai Tesque). Briefly, a 1/10 volume of WST-8 solution was added to each well, and the plates were incubated at 37 °C for 1 h. Then, the absorbance at 450 nm was measured in a microplate reader.

On a layer of 1% bottom agar (Sigma-Aldrich) 10000 NIH-3T3 cells per well of a 12-well plate were suspended in DMEM (Thermo Fisher Scientific) containing 0.6% agar, 10% calf serum (PAA Laboratories), 0.5% sodium bicarbonate (PAN Biotech) and 1% sodium pyruvate (Thermo Fisher Scientific). After 16 days incubation pictures were taken with a Zeiss Axiovert 40 CFL microscope at × 100 magnification and colony size was assessed with ImageJ (http://rsbweb.nih.gov/ij/).